Cell surface display of polypeptide isoforms by stop codon readthrough

Abstract

The application describes a method of selecting mammalian host cells that express a polypeptide of interest with high yield. The host cells contain an expression cassette with a first polynucleotide encoding a polypeptide of interest, at least one leaky stop codon located downstream of the first polynucleotide and a second polynucleotide located downstream of the leaky codon encoding an immunoglobulin transmembrane anchor comprising a cytoplasmic domain. The host cells are cultivated to allow expression of the polypeptide of interest such that some of the polypeptides of interest are expressed as fusion proteins displayed on the cell surface. High producing cells are then selected based on the presence or amount of the displayed fusion polypeptides.

Claims

1. A method for producing a polypeptide of interest with high yield, the method comprising: a) providing a plurality of mammalian host cells comprising a heterologous nucleic acid comprising at least one cassette (Cas-POI) comprising a first polynucleotide (Pn-POI) encoding the polypeptide of interest, at least one leaky stop codon downstream of the first polynucleotide, and a second polynucleotide downstream of the leaky stop codon encoding an immunoglobulin transmembrane anchor comprising a cytoplasmic domain; b) cultivating the mammalian host cells to allow expression of the polypeptide of interest such that at least a portion of the polypeptide of interest is expressed as a fusion polypeptide comprising the immunoglobulin transmembrane anchor directly anchored to the cell membrane comprising a cytoplasmic domain, wherein at least a portion of said fusion polypeptide comprising the immunoglobulin transmembrane anchor is displayed on the surface of said mammalian host cells to allow binding of a detection compound; c) selecting at least one of the plurality of mammalian host cells based upon to the presence or amount of the fusion polypeptide displayed on the cell surface; d) culturing the selected mammalian host cell in culture medium under conditions that allow for expression of the polypeptide of interest wherein the polypeptide of interest is secreted into the culture medium; and e) obtaining the polypeptide of interest from the culture medium.

2. The method according to claim 1, wherein the immunoglobulin transmembrane anchor is selected from the group consisting of a) a transmembrane anchor derived from IgA, IgE, IgM, IgG and/or IgD, and b) an immunoglobulin transmembrane anchor comprising a sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 7.

3. The method according to claim 1, wherein step c) comprises contacting the plurality of mammalian host cells with a detection compound binding the fusion polypeptide and selecting at least one mammalian host cell based upon the presence or amount of the bound detection compound.

4. The method according to claim 1, wherein two or more selection cycles are performed, wherein in each selection cycle at least one mammalian host cell is selected based upon the presence or amount of the fusion polypeptide displayed on the cell surface.

5. The method according to claim 3, wherein binding of the detection compound to the surface of the mammalian host cell is detected by flow cytometry.

6. A method for producing a polypeptide of interest, comprising culturing a mammalian host cell which comprises at least one cassette comprising: a) at least a first polynucleotide encoding a polypeptide of interest; b) at least one leaky stop codon downstream of the first polynucleotide; and c) a second polynucleotide downstream of the leaky stop codon encoding an immunoglobulin transmembrane anchor comprising a cytoplasmic domain, wherein at least a portion of said immunoglobulin transmembrane anchor is displayed on the surface of said mammalian host cells to allow binding of an immunoglobulin.

7. The method for producing a polypeptide of interest according to claim 1, further comprising one or both of the following steps: a) purifying the expressed polypeptide; and b) further processing or modifying the expressed polypeptide.

8. The method for producing a polypeptide of interest according to claim 6, further comprising at least one step selected from the steps: obtaining the polypeptide from the cell culture; obtaining the polypeptide from the culture medium wherein the polypeptide is secreted into the culture medium; isolating the expressed polypeptide; purifying the expressed polypeptide; and further processing or modifying the expressed polypeptide.

Description

EXAMPLES

Example 1

Vector Construction of the Ig Transmembrane Version

(1) A synthetic 1113 bp DNA fragment encoding part of the IgG1 constant heavy chain region plus the leaky stop codon stuffer and the Ig transmembrane and cytoplasmic domain is inserted into pBW201 (a standard vector containing an IgG1 heavy chain and kappa light chain) via Age1 and Asc1 generating pNT11 (see table 1). The nucleotide sequence of the Ig transmembrane domain used is shown in SEQ ID No: 1, the leaky stop codon stuffer is indicated. Of course, also variants of the encoded Ig transmembrane domain can be used according to the principles of the present invention which provide the same membrane anchoring function. Said variants are homologue to the encoded Ig transmembrane domain and can e.g. be obtained by conservative amino acid substitution. They share preferably at least 80%, 85%, 90% homology. Polynucleotides encoding respective variants e.g. hybridize to the shown sequence under stringent conditions.

(2) The wt DHFR selection marker gene of pNT11 and pBW201 can be replaced by a synthetic 1252 bp fragment encoding a L23P point mutant of DHFR via Swal and BgIII, thereby generating pNT29 and pBW478. The DHFR mutant allows the selection of dhfr+ cell lines.

(3) The FACS vectors (pNT11, pNT29) are based on the standard vectors for antibody expression (pBW201, pBW478). pNT11 and pBW201 are differing from pNT29 and pBW478 in the DHFR selection marker cassette they are carrying. Apart from that the backbones are identical. The vector has a mono-cistronic “tandem” setup and contains antibody light and heavy chain expression cassettes, both driven by the CMV promoter/enhancer. The only modification to generate the FACS vectors was the insertion of an IgG1 transmembrane and cytoplasmic domain 3′ of the antibody heavy chain (HC) cDNA. A short stuffer with a leaky translation termination signal is placed between HC and transmembrane domain. The sequence environment selected for the stop codon is expected to lead to a read-through of up to 5%. All four vectors are coding for the same human IgG antibody.

(4) As is outlined above, the vector nucleic acids used for expression and in particular the orientation and arrangement of the vector elements chosen allow the very efficient expression of immunoglobulin molecules. Suitable vectors that can be used in conjunction with the present invention and which are described above are illustrated in the following table (the arrows indicate the 5′ to 3′ orientation of the genetic elements):

(5) TABLE-US-00001 TABLE 1 Vector map pNT11 - “FACS vector” CMVprom/enhan .fwdarw. RK-intron .fwdarw. mAB-LC .fwdarw. SV40polyA .fwdarw. CMV prom/enhan .fwdarw. RK-intron .fwdarw. mAB-HC .fwdarw. Stuffer + leaky stop codon Ig transmembrane domain and cyto-plasmatic domain .fwdarw. SV40polyA .fwdarw. Phage f1 region .fwdarw. SV40prom/enhan .fwdarw. Neo .fwdarw. Synth polyA Amp .fwdarw. SV40prom/enhan .fwdarw. DHFR .fwdarw. SV40pA .fwdarw.

(6) The abbreviations in table 1 have the regular meaning as apparent for the person of skill in the art and as described above, and have in particular the following meanings: CMVprom/enh=human cytomegalovirus immediate early promoter/enhancer RK-intron=comprises the intron donor splice site of the CMV promoter and the acceptor splice site of the mouse IgG Heavy chain variable region (see e.g. Eaton et al., 1986, Biochemistry 25, 8343-8347, Neuberger et al., 1983, EMBO J. 2(8), 1373-1378; it can be obtained from the pRK-5 vector (BD PharMingen)) mAB-LC=monoclonal antibody light chain mAB-HC=monoclonal antibody heavy chain SV40polyA=SV40 polyA site SV40prom/enhan=SV40 promotor/enhancer Neo=neomycin phosphotransferase Synth polyA=synthetic polyadenylation site Amp=beta lactamase antibiotic resistance gene DHFR=dihydrofolate reductase gene.

Example 2

Transfection and Selection of CHO-Cells

(7) Cell cultivation, transfection and screening is carried out in shake flasks using suspension growing CHO cells in a proprietary, chemically defined culture medium. Cells are either transfected by lipofection or electroporation (nucleofection) following the manufacturer's instructions. Transfection efficiency is checked by transfecting a GFP (green fluorescence protein)-reporter plasmid and flow cytometric analysis of the transfected cells. Depending on the cell viability, selection is started 24-48 h after transfection by adding G418 containing selective medium to the cells. As soon as cells recover to a viability of above 80%, a second selection step is applied by passaging the cells to G418 free, MTX (methotrexate) containing medium. After recovery of the cells from the MTX selection, cultivation is continued in MTX containing medium throughout FACS enrichment cycles, FACS cloning or limited dilution cloning and screening.

(8) Cell viability and growth are monitored using an automated system (ViCell, Beckmann Coulter).

Example 3

FACS Analysis, Enrichment and Cloning of Cells

(9) Labeling of cells: 2×10E7 cells per transfected pool are centrifuged and washed with 5 mL of chilled PBS (phosphate buffered saline) and resuspended in 1 mL of cold PBS. A suitable amount of FITC (fluorescein isothiocyanate) labeled anti-IgG antibody is added to the cells and is incubated on ice for 30 minutes in the dark. Subsequently, cells are washed twice at room temperature with 5 mL PBS, resuspended in 1 mL PBS, filtrated and dispensed into a FACS tube for analysis, sorting and cloning.

(10) Analysis, sorting and cloning of cells: The cell sorting is performed with a FACSAria (Becton Dickinson) equipped with an Automatic Cell Deposition Unit (ACDU) using FACSDiva software. A low powered air-cooled and solid-state laser (Coherent® Sapphire™ solide state) tuned to 488 nm is used to excite fluorescein dyes bound to the secondary antibody. The relative FITC fluorescence intensity is measured on E detector through a 530/30 BP filter. Five percents of the highest FITC fluorescent cells are gated and sorted either in block or as single cells in 96 well plates.

Example 4

Determination of Clonal Productivity and Stability

(11) Productivity of clones is analyzed in batch and fed batch experiments using different formats. Initial clone screening is performed in 24-well plate batch assays by seeding cells to shaken 24-well plates. Antibody concentrations in the cell culture supernatant are determined by protein-A HPLC 10d after starting the culture. The highest producing clones are also analyzed in shake flask models in batch and fed batch mode. Batch cultures are seeded in shake flask 500 with 100 mL working volume and are cultivated in a shaker cabinet (not humidified) at 150 rpm and 10% CO2. Viability of cells should be >90% when starting the assay. The seeding cell density is 2×10.sup.5 c/mL. Product concentration/cell number/viability determination took place at day 3-7, 10 and 13. Fed batch experiments are done using the same conditions but with a starting cell density of 4×10.sup.5 c/mL and with regular adding of feeds. Clonal stability is evaluated by culturing the cells over a period of 14 weeks with productivity measurements using the shake flask batch model every two weeks.

Example 5

Analysis of Transiently Transfected Cells

(12) To test whether membrane bound translation products are present on the cell surface after transfection with the new FACS vector (here pNT11 or pNT29), transiently transfected cells are analyzed by immunostaining and flow cytometry. 48 h after transfection, cells are stained with a FITC-labeled antibody directed against human IgG. Cells transfected with a GFP expression vector are used as a transfection control, the transfection efficiency is calculated to be about 60%. Un-transfected cells and cells transfected with the standard vector (not comprising a transmembrane domain) do not show significant levels of surface associated antibody, while 16% of the cells transfected with the FACS vector are stained above background level. This shows that the fusion peptide, here an antibody molecule, anchored to the cell membrane can be detected on the cell surface.

Example 6

Analysis and Enrichment of Stable Transfected Cells

(13) Having shown presence of membrane bound antibody on transiently transfected cells the surface expression level and distribution in selected pools of transfected cells is analysed. Thereby, it can be shown that producing cells can be selectively enriched by FACS sorting. Therefore, cells after transfection are selected with G418 and subsequently with MTX. The resulting pools of resistant cells are stained with FITC labelled anti-IgG antibody and analyzed by flow-cytometry. As a control, un-transfected cells are stained and analyzed. Subpopulations of positive cells are detected in the selected pools transfected with the FACS vector. The distribution of positive cells thereby differed between the two analyzed pools. To assess whether high producing cells can be enriched based on their fluorescence signal (and hence allow a quantitative selection), cells having the highest fluorescence intensity are sorted (top 5%) from each of the two pools and sub-cultured to compare the productivity with the pool before enrichment.

Example 7

Analysis of Productivity of Enriched and Non Enriched Cells

(14) Productivity analyses of the selected pools before and after flow-cytometry enrichment are done in shake flask batch cultures to compare the end-product concentration at day 13. At day 13 the supernatant is harvested and analyzed for IgG content by Protein-A-HPLC. Both pools show a significant increase of production level already after performing one FACS enrichment cycle according to the teachings of the present invention. While product concentration for pool 1 increases by a factor of approximately 2, pool 2 increases by a factor of almost 10 showing that high producing cells are selectively detected during staining and sorting.

(15) Already in the first enrichment cycle antibody concentrations of almost 250 mg/l can be obtained.

Example 8

Flow-Cytrometry Based Selective Cloning of High Producing Cells

(16) Flow-cytrometry can be used to sort and seed individual stained cells according to their staining profile. To analyze whether such selective cloning results in higher number of high producing clones than cloning by limiting dilution, clones are generated using both methods and productivity is analyzed in 24-well plate batch cultures. Batch cultures in 24-well plates are done and at day 10 supernatants are harvested and measured for IgG content by Protein-A-HPLC. The results are as follows:

(17) TABLE-US-00002 TABLE 2 FACS sorting versus limited dilution (LD) 0-25 26-50 51-75 76-100 101-125 126-150 Method mg/l mg/l mg/l mg/l mg/l mg/l LD - obtained 12 0 1 0 1 0 clones FACS - obtained 2 2 2 2 0 1 clones

(18) The flow-cytrometry derived clones have a higher average productivity compared to the liming dilution derived clones, which is also reflected in the clonal distribution of the productivity range.

Example 9

Comparison of FACS and Standard Vector

(19) To confirm the beneficial effect of flow-cytometry enrichment of transfected cells and to compare use of the FACS vector (pNT29) with a standard vector, cells are transfected and selected with G418 and MTX. Three cell pools transfected with the FACS vector (samples 1, 2 and 3) and three cell pools transfected with the standard vector (samples 7, 9 and 9) are analyzed by flow cytrometry and the 5% having the highest staining signal are sorted. Shake flask batch cultures are done to compare the increase of product concentration after enrichment. Transfected and selected pools are stained and sorted by flow-cytrometry to enrich the top 5% based on the fluorescence intensity. Before and after enrichment, shake flask batch cultures are done and after 13 days supernatants are analyzed by Protein-A-HPLC. The results are as follows (approximately):

(20) TABLE-US-00003 TABLE 3 Results obtained with the FACS vector Sample Product concentration Sample 1; 10 mg/l   FACS vector, before enrichment Sample 1; 55 mg/ml FACS vector, 1st enrichment Sample 1; 100 mg/ml  FACS 2nd enrichment Sample 1; 365 mg/ml  FACS vector, 3rd enrichment Sample 2; 40 mg/ml FACS vector, before enrichment Sample 2; 65 mg/ml FACS vector, 1st enrichment Sample 2; 90 mg/ml FACS vector, 2nd enrichment Sample 2; 340 mg/ml  FACS vector, 3rd enrichment Sample 3; 15 mg/ml FACS vector, before enrichment Sample 3; 95 mg/ml FACS vector, 1st enrichment Sample 3; 155 mg/ml  FACS vector, 2nd enrichment Sample 3; 85 mg/ml FACS vector, 3rd enrichment

(21) TABLE-US-00004 TABLE 4 Results obtained with the Standard vector Sample Product concentration Sample 7; 40 mg/l   Standard vector, before enrichment Sample 7; 55 mg/ml Standard vector, 1st enrichment Sample 7; 50 mg/ml Standard 2nd enrichment Sample 7; 25 mg/ml Standard vector, 3rd enrichment Sample 8;  5 mg/ml Standard vector, before enrichment Sample 8; 10 mg/ml Standard vector, 1st enrichment Sample 8; 12 mg/ml Standard vector, 2nd enrichment Sample 8; 15 mg/ml Standard vector, 3rd enrichment Sample 9;  5 mg/ml Standard vector, before enrichment Sample 9;  2 mg/ml Standard vector, 1st enrichment Sample 9; 10 mg/ml Standard vector, 2nd enrichment Sample 9; 10 mg/ml Standard vector, 3rd enrichment

(22) As is demonstrated by the results, the production level of FACS vector transfected cells increases significantly for the tested three pools, while in case of the standard vector only one pool showed a significant increase in product concentration. The average of product concentrations after enrichment with the FACS vector is significantly higher as with the standard vector. Two further sequential FACS enrichment cycles are done to enrich high producing cells showing that only in case of the FACS vector productivity of the cell populations is increased. Finally, product concentrations can be increased by 4- to 30-fold.

(23) For comparison of the suitability of both vectors for selective cloning, clones from non-enriched pools with comparable productivity are selectively sorted by flow-cytometry. Subsequently, productivity of the clones is analyzed in 24-well batch cultures. Clones derived from FACS vector transfected pools are found to have a higher average expression level as clones from standard vector transfected pools. The clonal distribution of productivity shows that in case of the FACS vector a higher number of good producing clones is obtained (see table 5):

(24) TABLE-US-00005 TABLE 5 Standard vector versus FACS vector (pNT29) 0-50 51-100 101-150 151-200 201-250 251-300 301-350 Method mg/l mg/l mg/l mg/l mg/l mg/l mg/ml Stan- 31 7 0 2 0 1 0 dard vector FACS 21 20 4 4 4 2 1 vector

Example 10

Further Comparisons Between the FACS Vector and Standard Expression Vectors

(25) a) Vector Construction

(26) The vectors pBW201, pNT11, pBW478 and pNT29 are obtained as described in example 1.

(27) b) Transfection, Selection and Cloning of CHO Cells

(28) This is done as described in example 2.

(29) c) FACS Analysis, Enrichment and Cloning of Cells

(30) This is done as described in example 3.

(31) d) Determination of Antibody Production and Clonal Stability

(32) The productivity of clones and pools is analyzed in batch and fed batch experiments using different formats. Pools before and after FACS enrichment are analyzed in shake flask batch assays by seeding 1×10.sup.5 cells per mL (c/mL) in 50 mL working volume using shake flasks with 250 mL capacity. IgG content is analyzed by Protein-A HPLC from samples taken at day 13 of the batch culture. Initial screening of clones is performed in 24-well plate batch assays by seeding cells into shaken 24-well plates. Antibody concentrations in the cell culture supernatant are determined by quantitative Protein A-HPLC 10 days after starting the culture. The highest producing clones are analyzed in shake flask models in batch and fed batch mode. Batch cultures are seeded into shake flasks (500 mL capacity) with 100 mL working volume and are cultivated in a shaker cabinet (not humidified) at 150 rpm, 36.5° C. and 10% CO.sub.2. Viability of cells is >90% when starting the assay. The seeding cell density is 2×10.sup.5 c/mL. Antibody concentrations, cell number and viability are determined on days 3-7, 10 and 13. Fed batch experiments are done using the same conditions but with a runtime of 17 days and with a starting cell density of 4×10.sup.5 c/mL and with regular addition of feeds starting at viable cell densities above 7×10.sup.6 c/mL. Clonal stability is evaluated by culturing the cells over a period of 12 weeks with productivity measurements using the shake flask batch model every two weeks.

(33) e) Analysis and Enrichment of Stable Transfected Cells

(34) The surface expression in stably transfected cell populations is analysed to test whether producing cells can be selectively enriched by FACS-sorting. Therefore, cells after transfection are selected with G418 and subsequently with MTX. The resulting pools (10 per vector) of resistant cells are stained as described above and analyzed by flow-cytometry. With the used staining protocol positive sub-populations of cells could be detected in both, pBW478 and pNT29 transfected cell pools. As expected, a higher proportion of FACS positive cells is found with the FACS vector.

(35) To show that high producing cells can be enriched based on their fluorescence signal, cells having the highest fluorescence intensity are sorted (top 5%) from the individual cell pools and sub-cultured to compare the productivity with the pool before enrichment. A second cycle of enrichment is performed after expansion and pooling of the one times sorted cell populations. The percentage of staining positive cells surprisingly increased most with the standard vector in the first enrichment cycle. FACS-vector transfected pools showed similar enrichment factors with the used staining protocol and generally, significant pool to pool variation was observed. After the second enrichment cycle, almost homogeneously FACS positive cell populations are obtained (see Table 6a and 6b).

(36) Table 6a and 6B: Average Staining Results and Productivities Before and after Sorting

(37) TABLE-US-00006 TABLE 6a FACS analysis of stained cells before and after FACS enrichment cycles % cells above background pBW478 (reference vector) pNT29 (FACS vector) FITC staining No FACS 1x FACS 2x FACS No FACS 1x FACS 2x FACS AVG 5.9 83.2 90.4 14.2 46.6 90.5 STDD 3.396403 15.80158 1.126795 8.974284 13.51148 1.422439 Table 6a: Transfected and selected cell pools were stained for surface IgG. Average percentage of cells stained above the level of untransfected cells is shown. Before enrichment a higher percentage of positive cells is found with the FACS vector. After the first enrichment cycle of the top 5%, the proportion of staining positive cells was highest with the standard vector. After the second enrichment cycle, greater that 90% of all cells were positive with both approaches. Abbreviations: AVG: Average and STDD: Standard deviation.

(38) TABLE-US-00007 TABLE 6b Productivities of in shake flask batch model before and after FACS enrichment cycles mAb pBW478 (reference vector) pNT29 (FACS vector) (mg/L) No FACS 1x FACS 2x FACS No FACS 1x FACS 2x FACS AVG 38.5 123.4 68.3 46.5 171.8 363 STDD 17.66509 101.8563 6.592926 15.30614 114.1936 70.19259 Table 6b: Productivity of cell pools is analyzed from shake flask batch cultures by Protein-A HPLC at day 13 of the culture. The first enrichment cycle led to a significant increase of productivity in both cases. After the second enrichment, only FACS vector transfected cell pools showed additional increase in productivity.
f) Analysis of Productivity of Enriched and Non Enriched Cells

(39) Productivity analysis of the selected pools before and after flow-cytometry enrichment are done in shake flask batch cultures to compare the end-titers at day 13. Productivity of the pools before enrichment is in a very comparable range for both vectors used. With the first enrichment cycle on the individual pools significant improvement of the average productivity is achieved with all approaches and again, there is substantial variation between individual pools (see Table 6b). Surprisingly, the productivity of the standard vector transfected pools is not higher compared to the FACS vector transfected ones although a much higher level of FACS staining positive cells was seen before. By sorting a second time from the pooled sorted cell populations, no further improvement of productivity is achieved with the standard vector. In contrast, a lower productivity is obtained although the FACS-staining result suggested that almost 100% of the cells should be producing antibody (see Table 6a). Productivity of FACS-vector transfected pools could be significantly improved by sorting a second time. The used FACS procedure leads to more selective enrichment of high producing cells with a productivity increase of about at least 8-fold compared to unsorted population and at least 2-fold compared to the pools after one sorting.

(40) g) Flow-Cytrometry Based Selective Cloning of High Producing Cells

(41) Flow-cytrometry can be used to sort and seed individual stained cells according to their staining profile. To analyze whether such selective cloning results in higher number of high producing clones when using the FACS-vector compared to the standard vector, clones are generated using both methods and productivity is analyzed in 24-well plate batch cultures.

(42) In a first round, cells are directly FACS-cloned from the MTX selected cell pools without any pre-enrichment step. Three pools per vector are chosen based on their staining profile. Clones are generated from the top 5% of the stained cell pools and in total about 500 clones are analyzed. While average productivity of clones with the reference vector was 39 mg/L, FACS-vector clones produced an average of 87 mg/L. As shown in Table 7a, this is also reflected by the clonal distribution which confirms that a much higher proportion of high producing clones is obtained with the FACS-vector. Interestingly, one out of the over 270 clones analyzed from the standard vector transfection had an almost 2-fold higher productivity compared to the others. This exceptional clone is designated LP. Identifying such a high producing cell clone with the standard vector setup in combination with a FACS screening procedure is thus generally possible. However, it is a very rare and thus lucky event. This is also the decisive difference to the selection process according to the teachings of the present invention. While the standard set up allows the selection of (very) high producers only in exceptional and thus rare cases, the method according to the present invention allows the selection of (very) high producers reproducibly and thus reliably.

(43) A second FACS-cloning experiment is performed starting from the 10 pooled populations per vector after the first enrichment cycle. This time approximately 240 clones are screened in 24-well batch cultures. Again, clones obtained with the FACS-vector have a much higher average productivity than the reference standard vector. No improvement compared to cloning without pre-enrichment is achieved with the reference vector at an average clone productivity of 40 mg/L. The LP clone was not identified again. In case of the FACS vector transfected clones, an average productivity of 275 mg/L was obtained with the used FACS method. The clonal distribution clearly demonstrates the superiority of the FACS-vector setup with regards to selective cloning of high producers (see Table 7b).

(44) Table 7a and 7b: Comparison of Productivity of Clones

(45) TABLE-US-00008 TABLE 7a 24-well-Batch - Clonal distribution pBW478 pNT29   0-50 mg/L 196 163  51-100 mg/L 70 22 101-150 mg/L 8 11 151-200 mg/L 2 5 201-250 mg/L 0 13 251-300 mg/L 2 9 301-350 mg/L 1 10 351-400 mg/L 0 6 401-450 mg/L 0 5 451-500 mg/L 0 2 501-550 mg/L 0 1 551-600 mg/L 1 0 Table 7a: Clones are generated by flow-cytrometry from the top 5% of the three stained cell pools with the highest percentage of staining positive cells after selection. For productivity assessment, batch cultures in 24-well plates were done and at day 10 supernatants were harvested and measured for IgG content by Protein-A-HPLC. Shown here is the clonal distribution of the productivity range. A significantly higher proportion of high producing clones is obtained when using the FACS vector (pNT29).

(46) TABLE-US-00009 TABLE 7b 24-well-Batch: FACS Cloning Pooled pools pBW478 pNT29   0-50 mg/L 102 22  51-100 mg/L 17 2 101-150 mg/L 13 5 151-200 mg/L 5 3 201-250 mg/L 2 7 251-300 mg/L 0 11 301-350 mg/L 0 11 351-400 mg/L 0 15 401-450 mg/L 0 9 451-500 mg/L 0 7 501-550 mg/L 0 7 551-600 mg/L 0 1 601-650 mg/L 0 3 Table 7b: Clones obtained by FACS cloning from the top 5% of stained combined pools after one enrichment cycle were analyzed. No benefit the from pre-enrichment was found for reference vector (pBW478) transfected cells, while in case of FACS vector (pNT29) transfected cells, pre-enrichment led to a significant reduction of non-producers and to an increase of the average productivity of clones.
h) Characterisation of Clones

(47) The LP clone derived from the standard vector as well as 10 high producing FACS-vector clones are expanded to shake flasks and tested in generic shake flask batch and fed-batch models to evaluate their manufacturing potential.

(48) Productivity in batch cultures is found to be about the same in the range of 1 g/L for all clones tested. Fed-batch productivities are also very comparable for all clones and in the range of 3.5-4 g/L (see Table 8). No significant difference in growth parameters are observed when comparing the FACS-vector transfected clones with reference vector transfected clones (LP and clones from previous experiments). Also, production stability is found to be high for the FACS-vector derived clones, only one out of 10 analyzed clones showed a drop of productivity greater than 25% after 12 weeks in culture which is a lower ratio of unstable clones as it was observed with the reference standard vector in previous experiments (data not shown).

(49) TABLE-US-00010 TABLE 8 Pool productivities: Fed batch shake flask (SF) model mAb (g/L) SF batch SF fed batch 1 = LP 1.1 4.3 2 0.9 3.7 3 1.0 3.9 4 1.0 3.8 5 1.0 3.8 6 1.0 3.9 7 1.2 3.3 8 1.0 3.8 9 0.9 3.6 10  1.0 3.7 11  0.9 3.3 Table 8: The highest producing clone obtained with the standard vector (pBW478) and 10 clones derived from the FACS vector (pNT29) are analyzed in batch and fed batch shake flask cultures. IgG content is analyzed by Protein-A HPLC at day 13 (batch cultures) or day 17 (fed batch cultures). All analyzed cones produce in a comparable range.

Example 11

Large Scale Production of Polypeptides with Transfected CHO Cells

(50) The production of polypeptides in large scale can be done for example in wave, glass or stainless steel bioreactors. For that purpose the cells are expanded, usually starting from a single frozen vial, for example a vial from a Master Cell Bank. The cells are thawed and expanded through several steps. Bioreactors of different scale are inoculated with appropriate amounts of cells. The cell density can be increased by adding feed solutions and additives to the bioreactor. Cells are kept at a high viability for a prolonged time. Product concentrations in the reactor ranging from a few hundred milligrams per litre up to several grams per litre are achieved in the large scale. Purification can be done by standard chromatography methodology, which can include affinity, ion exchange, hydrophobic interaction or size exclusion chromatography steps. The size of the bioreactor can be up to several thousand litres volume in the final scale (see also e.g. F. Wurm, Nature Biotechnology Vol. 22, 11, 2004, 1393-1398).